Dynamic Transcription Machineries Guide the Synthesis of Temporally Operating DNAzymes, Gated and Cascaded DNAzyme Catalysis

Transient transcription machineries play important roles in the dynamic modulation of gene expression and the sequestered regulation of cellular networks. The present study emulates such processes by designing artificial reaction modules consisting of transcription machineries that guide the transient synthesis of catalytic DNAzymes, the transient operation of gated DNAzymes, and the temporal activation of an intercommunicated DNAzyme cascade. The reaction modules rely on functional constituents that lead to the triggered activation of transcription machineries in the presence of the nucleoside triphosphates oligonucleotide fuel, yielding the transient formation and dissipative depletion of the intermediate DNAzyme(s) products. The kinetics of the transient DNAzyme networks are computationally simulated, allowing to predict and experimentally validate the performance of the systems under different auxiliary conditions. The study advances the field of systems chemistry by introducing transcription machinery-based networks for the dynamic control over transient catalysis—a primary step toward life-like cellular assemblies.


INTRODUCTION
Transient transcription machineries play important roles in the dynamic modulation of gene expression and sequestered regulation of cellular processes. 1−4 Spatial and temporal misregulation of gene expression programs leads to diverse diseases, and developing means to inhibit the misregulated transcription pathways is a scientific challenge. 5 Indeed, mimicking transient dynamic machineries by artificial means and the development of methods to block and control gene expression circuitries is a key goal of systems chemistry. 6,7 In addition, dynamic gene expression machineries play important roles in intercommunicating complex genetic networks and the fan-out or branched production of multifunctional proteins. 8−10 Accordingly, modulating such processes by synthetic means provides primary steps toward synthetic cell func-tions�protocells. 11,12 Recent advances in DNA nanotechnology used the information encoded in the base-sequence of the DNA biopolymer to assemble dynamic DNA circuitries and networks. 13−15 The dynamic features of these systems were guided by the structural reconfiguration of single-stranded nucleic acid or duplex DNA scaffolds. Different triggers were used to reconfigure DNA structures including strand displacement, 16,17 formation and dissociation of G-quadruplexes 18,19 or triplex structures, 20 and the use of light and photoisomerizable intercalators to stabilize/destabilize nucleic acid duplexes. 21 These triggered DNA reconfiguration motives were applied for the dynamic assembly of complex reaction circuitries and programmed network assemblies. 22,23 Dynamically triggered constitutional dynamic networks, revealing adaptive, 15,24 hierarchically adaptive, 25 feedback-driven, 26 intercommunicated features, 27 were reported and their use for programmed dynamic catalysis 28 were demonstrated. Particularly, transient out-of-equilibrium, dissipative nucleic-acid-based networks attracted substantial recent research efforts. 29−32 Enzymeguided transient networks driven by ligase, endonucleases, or nickases were reported, 33−35 and dynamic reaction circuits revealing oscillatory behaviors, 36,37 gated and cascaded transient operations 38−40 using dissipative reconfiguration of dynamic networks were achieved. Also, network-guided transient biocatalytic reaction modules dictating transient enzyme cascades, 41 light-induced formation and dissipative depletion of microscale structures, e.g., microtubules, 42,43 or nanoparticle aggregates, 44 and transient enzyme-guided release and uptake of loads were demonstrated. 45,46 The modulation of transcription machineries in nature by auxiliary triggers such as miRNA, plays important roles in gene expression. 47 The modularity of transcriptional circuits provides a "toolbox" of nucleic acid structures for the catalytic synthesis of dynamically modulated transcriptional frameworks and artificial circuits. 48−51 Indeed, transcriptional oscillators, 37,50 transcriptional switches, 52 and bistable gene-regulatory networks 53 were reported. Nonetheless, while recent efforts addressed the dynamic control over transcriptional circuits and transcription/translation networks using fuel/ antifuel strand displacement triggers, 54 enzyme-driven assembly/disassembly of DNA-RNA nanotubes 55,56 or colloidal nanoparticles, 57 and the autoinhibited transcription of RNA in the presence of RNA polymerase aptamer as inhibitor, 58 the transient, triggered activation of temporally operating catalytic under different auxiliary conditions. Dots (denoted by x) in different panels correspond to experimental data, and solid curves (denoted by x′) correspond to computationally simulated data. Panel I: a/a′, experimental and computational conditions as presented in (C); b′, computationally simulated data using the set of reaction rates derived for curve a′ but using NTPs = 1 mM; b, validated experimental results in the presence of NTPs = 1 mM. All other conditions are as described in (C). Panel II: b/b′ as stated in panel I; c′, computationally simulated data using the set of reaction rates derived for curve a′, c, validated experimental results, in the presence of NTPs = 1 mM, RNase H = 7 U/mL (0.233 nM); d′, computationally simulated data, d, validated experimental results, in the presence of NTPs = 1 mM, RNase H = 8 U/mL (0.266 nM). All other conditions are as described in (C). Panel III: d/d′ as stated in panel II; e′, computationally simulated data using the set of reaction rates derived for curve a′, e, validated experimental results, in the presence of NTPs = 1 mM, T7 RNAP = 3 U/μL (0.048 μM), RNase H = 8 U/mL (0.266 nM); f′, computationally simulated data, f, validated experimental results, in the presence of NTPs = 1 mM, T7 RNAP = 4 U/μL (0.064 μM), RNase H = 8 U/mL (0.266 nM). All other conditions are the same as those stated in (C). Error bars represent standard deviations of three measurements. agents and chemical transformations driven by transcriptional machineries is a challenging topic that needs to be addressed.
In the present study, we introduce a reaction module in which the nucleoside triphosphates (NTPs) fueled, triggered activation of a transcription machinery leads to the transient temporal formation of Mg 2+ -ion-dependent DNAzyme catalysts. The integration of RNase H in the system, which specifically cleaves RNA hybridized to DNA, leads to the dissipative depletion of the DNAzyme catalyst at the expense of "waste" products generated by the degradation of the transcribed RNA. By treatment of synthetically designed reaction modules with appropriate blockers, gated selective temporal operation of one of two dictated DNAzymes is demonstrated, and by coupling of two transcription machineries, the temporal cascaded transient operation of two dissipative DNAzymes is introduced. Previous reports discussed the application of DNAzymes as functional constituents to operate the transient reaction modules. 39,40 Also, the transient synthesis of DNAzyme products by applying nucleic acid reaction modules, fuel-strands and nickase, 38 or ATP-driven ligation and nickase 35 as operators guiding the transient behavior of the reaction modules were demonstrated. The integration of the transcription machinery as a functional vehicle that drives the transient operation of DNAzyme would, however, advance the plethora of transcription-stimulated processes mimicking natural processes. The present study introduces the application of transcription machinery coupled to RNase as a mechanistic cycle for the transient operation of DNAzymes. Particularly, the transcription machinery/RNase system allows us to develop transient operating gated and cascaded DNAzyme networks as a primary model step toward life-like cellular transformations. The different dynamic reaction modules involving the transcription machinery-guided temporal synthesis and separation of the DNAzymes are accompanied by kinetic models that account for the kinetic behaviors of the transient networks. Computational simulations of the experimental results by the kinetic models not only provide a means to evaluate the rate constants of the subreactions involved in the dynamic operation of the systems but also allow to predict and experimentally validate the kinetic behaviors of the systems under different auxiliary conditions.

RESULTS AND DISCUSSION
A Nucleotide Fuel Mixture Activates the Transcription Machinery to Guide the Synthesis of a Transiently Operating DNAzyme. The transient synthesis of a Mg 2+ -ion-dependent DNAzyme by a transcription machinery is schematically outlined in Figure 1A. The reaction module consists of a transcription template consisting of a promoter strand N 1 hybridized with two strands P 1 and T 1 , a functional duplex composed of M 1 /L 1 , where M 1 corresponds to a subunit of the Mg 2+ -ion-dependent DNAzyme α, a single strand M 2 that corresponds to the second subunit of the DNAzyme, T7 RNA polymerase (RNAP), and RNase H. Within this configuration, caging M 1 in the duplex structure M 1 /L 1 prohibits the assembly of the DNAzyme structure. Subjecting the reaction module to the NTPs (as fuel) activates the transcription machinery that synthesizes RNA (R 1 ). The resulting transcribed R 1 is engineered to displace the duplex M 1 /L 1 to yield R 1 /L 1 and to release strand M 1 that selfassembles into the M 1 /M 2 DNAzyme structure. The resulting duplex R 1 /L 1 is cleaved, however, by RNase H to release L 1 that separates the intermediate Mg 2+ -ion-dependent DNA-zyme, leading to the regeneration of the rest reaction module of the system. The activation of the transcription machinery in the reaction module with the NTPs fuel leads to the R 1stimulated transient synthesis of the Mg 2+ -ion-dependent DNAzyme intermediate that is dissipatively depleted by RNase H to regenerate the rest reaction module. By extruding samples from the dynamic reaction system, the transient formation and depletion of the Mg 2+ -ion-dependent DNAzyme α can be quantitatively probed by following the kinetics of the DNAzyme-stimulated cleavage of the fluorophore/quenchermodified substrate S 1 . Figure 1B schematically depicts the transient kinetic formation and dissipative depletion of the intermediate DNAzyme driven by the NTPs-fueled transcription machinery and the accompanying RNase "waste" generating process that degrades the intermediate DNAzyme. Figure Figure S2B). Furthermore, it should be noted that RNase H does not affect the DNAzyme substrate 59 (see Figure S3). (Also, for additional optimization of the system shown in Figure 1, the temporal reaction module was probed at additional auxiliary conditions displayed in Figure S4.) To account for the experimental transient behavior of the formation and dissipative depletion of the DNAzyme, we formulated a kinetic model that includes the set of subreactions participating in the transient process ( Figure S5), and the experimental results were computationally simulated following the kinetic model. The computationally simulated transient behavior of the DNAzyme is displayed in Figure 1C, curve a′, overlaid on the experimental results. (In order to simulate the curves, a set of background experiments elucidating rate constants related to the kinetics and aimed to guide the effective simulation process were essential. This set of experiments and their significance were addressed in Figures S6−S11 and the accompanying discussions explaining these experiments, Supporting Information, pages S9−S18.) The set of computationally derived rate constants of the subreactions comprising the kinetic model are summarized in Table S1. (The sets of computationally derived rate constants of the background experiments are summarized in Tables S2 and S3). The kinetic model and the derived computationally simulated rate constants are of scientific value only if they have a predictive power on the kinetic behavior of the system under different auxiliary conditions that can be, subsequently, validated experimentally. Accordingly, we applied different auxiliary conditions to the transient appearance and depletion of the intermediate DNAzyme and probed the predicted transient behavior of the DNAzyme by applying the kinetic model and the derived set of simulated rate constants. We then validated the predicted kinetic behavior by experiments. The temporal concentrations of the DNAzyme were evaluated at the new auxiliary conditions ( Figure S12). Figure 1D, panel I, solid line, depicts the computationally predicted transient behavior of the DNAzyme at the NTPs concentration of 1 mM (the set of rate constants were derived for NTPs concentration corresponding to 0.5 mM, curve a′). The dotted curve b depicts the experimentally validated results. Very good agreement between the predicted and experimentally validated results is observed. Figure 1D, panel II depicts, in curve b/b′, the computational and experimental results of the system at NTPs concentration of 1.0 mM and RNase H, 6 U/mL. The set of derived rate constants was applied to predict the transient behavior of the system at a RNase H concentration corresponding to 7 U/mL and 8 U/mL, respectively. The predicted transient results are displayed in panel II, curves c′ and d′. The predicted transient behaviors at these conditions were, then, validated and presented in curves c and d (dotted curves). As before, the experimental results overlay nicely the predicted computational results. Figure 1D, panel III, shows the computationally predicted transient behavior of the DNAzyme using the set of derived rate constants in the presence of T7 RNA polymerase, 2 U/μL, curve d′, 3 U/μL, curve e′, and 4 U/μL, curve f′, respectively, and the dotted curves correspond to the experimentally validated results, curves d, e, and f, respectively. The formulated kinetic model and the derived computationally simulated rate constants provide a solid computational framework to quantitatively predict the transient behavior

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www.acsnano.org Article formation of the bifunctional duplex R 3 /L 1 +L 3 and the release of the M 1 and M 3 strands. While the released strands M 1 and M 3 self-assemble into two Mg 2+ -ion-dependent DNAzymes, M 1 /M 2 , M 3 /M 4 , the resulting R 3 /L 1 +L 3 is "digested" by RNase H to yield the R 3 "waste" products and the separated strands L 1 and L 3 . The latter two strands separate the transient DNAzyme structures to recover the rest reaction module. Thus, the NTPs-fueled activation of the reaction module activates the transcription machinery that yields two transient DNAzyme intermediates that are depleted by the degradation of the transcribed RNA. The transient formation of the system was then quantitatively probed by the cleavage of the substrates F 1 /Q 1 -modified substrate S 1 and the F 2 /Q 2modified substrate S 2 , activated by the two DNAzymes α and β ( Figure S13). Figure Tables S4−S6. The significance of these sets of rate constants rests on the ability to predict the gating efficiency of the system at variable concentrations of the blockers (for example, see Figure S17).

A Transient Catalytic DNAzyme Cascade Operated by Interconnected Dynamic Transcription Machineries.
The transcription machineries were further applied to operate a transient catalytic DNAzyme cascade ( Figure 4A). The system consists of two reaction modules. Module I includes the transcription template N 4 /T 4 , the caged duplex M 3 /L 3 , and the free single strand M 4 , where M 3 and M 4 correspond to the subunits of DNAzyme β. The enzymes T7 RNAP and RNase H are also included in the reaction module. In addition, a duplex P 1 /Q 1 is included in the module, and it acts as a key functional element that intercommunicates between modules I and II. Module II includes an incomplete, inactive, transcription template, N 1 /T 1 , the caged duplex M 1 /L 1 , and the single strand M 2 , where M 1 and M 2 correspond to the subunits of DNAzyme α, and the enzyme T7 RNAP and RNase H. In

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www.acsnano.org Article regenerates the inactive template N 1 /T 1 associated with module II, and together with the transient, generated M 1 /L 1 duplex restores the rest module II, and completes the recovery of the rest module I. The conjugation of the two modules leads to the activation of the formation of a transiently operating cascade of two DNAzymes, DNAzyme β and DNAzyme α. The transient operation of the two DNAzymes was then probed by sampling the catalytic activities of DNAzyme β and DNAzyme α along the temporal operation of the cascaded catalytic system. Figure 4B depicts the time-dependent fluorescence changes corresponding to the rates of cleavage of substrate S 2 by DNAzyme β, panel I, and the rates of cleavage of substrate S 1 by DNAzyme α, panel II, from samples extruded at time intervals from the two-module reaction mixture. Using appropriate calibration curves relating to the rates of cleavage of substrates S 1 and S 2 by variable concentrations of the DNAzymes (Figure S1), quantitative temporal concentrations of the DNAzymes were assessed, and these are displayed in Figure 4C, curves i and ii, respectively. The two cascaded DNAzymes reveal transient formation and depletion behaviors. The kinetics of the two-enzyme cascade was computationally simulated. A kinetic model for the system was formulated ( Figure S18) where additional subreactions associated with the coupling between the two moduli were integrated into the set of subreactions associated with DNAzyme β and DNAzyme α. Using the kinetic model, the computationally simulated kinetic behavior of the DNAzymes are overlaid the experimental results, curves i′ and ii'′ (solid curves, Figure 4C). The derived rate constants for the set of subreactions associated with the cascaded network are summarized in Table S7. Several important features regarding the cascaded temporal kinetics of DNAzyme β and DNAzyme α should be mentioned: (i) The activation of module II by the module I-generated promoter P 1 suggests that the temporal accumulation of P 1 will introduce a lag time interval in the transient operation of DNAzyme α generated by the transcription machinery of module II. Indeed, the inset of Figure 4C depicts the temporal kinetics of DNAzyme β and DNAzyme α at a short time interval of the temporal operation of the two DNAzymes. Obviously, DNAzyme α shows a short lag period of ca. 20 min and a delay in its peak content as compared to DNAzyme β. This result is consistent with the temporal accumulation of P 1 for operating module II. (ii) The cascaded operation of DNAzyme β and DNAzyme α shows a higher temporal activity of DNAzyme β as compared to DNAzyme α. Furthermore, the activity of DNAzyme α in the cascaded process is lower than the temporal activity of DNAzyme α in the single module construct ( Figure 1C, panel II). Two reasons might cooperatively contribute to the lower temporal activity of DNAzyme α. One reason is associated with the supply of promoter P 1 by module I to activate the transcription machinery of module II. As the temporal operation of module I involves the RNase H depletion of P 1 , the supply of P 1 to module II is dampened by the temporal behavior of module I. A second reason for the lower activity of DNAzyme α in the cascaded system might originate from the higher concentration of RNase H used to operate the cascaded system (10 U/mL) as compared to the concentration of RNase H in the single module system generating DNAzyme α ( Figure 1C, 6 U/mL). The higher concentration of RNase H depletes the intermediate DNAzyme α faster, leading to a lower content of this catalyst. A further control experiment involving a modified template in module I has emphasized the key interrelationship between modules I and II that is needed to operate the two-layer cascade (for explanation of this control experiment, see Figure S19 and accompanying discussion, and for computationally simulated kinetic model and the derived rate constants, see Figure S20 and Table S8).

CONCLUSION
The study has introduced a versatile concept to emulate native networks where transient transcription machineries modulate transient, dissipative, cellular processes. The concept is based on the design of reaction modules consisting of transcription templates, caged and inactive nucleic acid constituents exhibiting structural ability to assemble into catalytically active DNAzyme structures. The reaction modules include two enzymes, T7 RNAP and RNase H, as catalytic agents that control the dynamic modulation of the reaction modules. In the presence of the nucleotide bases, NTPs, as fuel, the transcription machineries are activated toward the RNAPcatalyzed synthesis of intermediate products that displace the caged constituents and allow the self-assembly of catalytically active Mg 2+ -ion-dependent DNAzyme units. The concomitant RNase-stimulated digestion of the RNA products releases, however, the free caging constituents, resulting in the dissociation of the self-assembled DNAzyme units and the recovery of the parent muted reaction modules. Accordingly, the systems mimic biological dissipative reaction pathways where the energy input�the NTPs fuel�activates the transient formation of intermediate catalysts, through the transcription-guided synthesis of RNA, that is degraded into "waste" products while allowing the temporal operation of the DNAzyme catalysts. The transient operation of the DNAyzmes is probed by the temporal DNAzyme-catalyzed cleavage of the respective fluorophore/quencher-functionalized substrates. By designing different reaction modules, gated, transcription machinery-guided transient operation of two different DNAzymes is demonstrated. In addition, by intercommunication of two functional reaction modules, the transcriptionguided cascaded transient operation of two DNAzymes is accomplished. In fact, recent reports discussed different methods to modulate transcription machineries by artificial means. These included the temporal transcription-guided assembly and RNase-induced disassembly of microtubes 55 or aggregated nanoparticles, 57 the transcription-controlled temporal ligation and nicking of intercommunicated DNA assemblies, 60 and the transcription machinery temporal modulated synthesis of a ribozyme through the transient inhibition of T7 RNAP by a transcribed aptamer. 58 Thus, the advantages and advancements introduced by the present study should be addressed. While the RNase-modulated transcription-controlled formation and depletion of DNA microstructures or particle aggregates 55,57,60 did not lead to any temporal catalytic outputs, only the transcription-inhibited T7 RNAP system led to a catalytic ribozyme output. 58 Nonetheless, in this later system, the transient formed ribozyme is accumulated in the system, in contrast to the transcription/ RNase-modulated depletion of the DNAzymes formed in the present study. The depletion of the transcription-modulated DNAzymes is particularly important in view of the envisaged medical applications of such transient DNAzyme agents. The transient formation and depletion of DNAzymes is envisaged to act as a temporal treatment agent of a medical event, and thus the accumulation of the catalyst should be prevented.

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Beyond the combined experimental and computational kinetically modeled transcription circuitries presented in our study, our results contribute a possible future medical application of the systems. An important facet of the study is the kinetic modeling of the complex dissipative transformations. The formulation of kinetic models and the computational simulation of the rate constants of the subreactions involved in these temporal dissipative systems is suggested as a key step for understanding the systems and as versatile method that should be adopted to quantitatively analyze dynamic networks. The computational simulations of the kinetics of dynamic networks not only provide rate constants that quantitatively account for the subreactions associated with the networks but also introduce tools to predict the behavior of the networks under different auxiliary conditions that can be later experimentally validated.
Probing the Activity of Transiently Operating DNAzymes. For each transcription machinery-guided dissipative DNAzyme system prepared, aliquots of 100 μL were withdrawn at time intervals and treated with 1 μL of fluorophore/quencher-modified substrate S 1 or S 2 stock solution (100 μM). The time-dependent fluorescence changes (λ ex = 496 nm, λ em = 520 nm) generated by the cleavage of FAM/BHQ1-modified substrate S 1 by DNAzyme α and/or fluorescence changes (λ ex = 588 nm, λ em = 608 nm) generated by the cleavage of ROX/BHQ2-modified substrate S 2 by DNAzyme β were monitored on a Cary Eclipse Fluorometer (Varian Inc.) at 25°C using plastic cuvettes with 10 mm path lengths. The temporal concentrations of the DNAzymes were quantified by using the appropriate calibration curves relating to the catalytic rates (d(ΔF)/ dt) of cleavage of substrates by variable standard concentrations of Mg 2+ -ion-dependent DNAzymes.
Chemicals, oligonucleotide sequences, transcriptionguided synthesis of malachite green aptamers, real-time fluorescence monitoring of the RNA transcript-mediated strand displacement, calibration curves corresponding to DNAzyme activities, DNAzyme activities in the absence of NTPs fuels, operation of two cycles of the DNAzyme α system, the effect of RNase H on DNAzyme substrate, optimization of the DNA template concentrations, computational kinetic models and discussions for simulations of the transcription machineries-guided synthesis of the temporally operating DNAzymes, gated and cascaded DNAzyme catalysis, control experiment for the two-layer cascaded DNAzyme system, tables summarizing the rate constants derived from the computational simulation (PDF)